Enveloped viruses enter and infect target cells by fusing their membrane envelope with the cytoplasmic or an internal membrane of the target cell. The spike glycoproteins of viral envelopes provide the best known biological fusion factors, and influenza hemagglutinin (HA) and some other spike glycoproteins constitute the best available systems for analyzing the process of biological fusion. Yet, the mechanism of viral spike glycoprotein-mediated membrane fusion is not well understood. Prior to fusion, spike glycoproteins undergo a conformational change, which involves the exposure of a rather hydrophobic "fusion peptide". It is generally assumed, but has never been directly proven, that fusion is triggered by the insertion of one or more fusion peptide(s) into the cellular target membrane. The goal of this proposal is to provide a detailed mechanism by which the viral spike glycoproteins of the influenza and vesicular stomatitis viruses, HA and G, respectively, catalyze the fusion of these viral envelopes with their target membranes. The specific aims are: (1) to provide direct (in situ) demonstration of the insertion of the fusion peptides of HA2 and G into their respective target membranes; (2) to determine whether fusion involves the cooperative action of several spike glycoproteins; (3) to determine whether "non-bilayer" lipids are directly involved in the fusion process; and (4) to define the structural requirements for the HA2-fusion peptide which promote membrane fusion. These aims will be accomplished by taking a biophysical approach, which combines the supported planar membrane model system with fluorescence micro-spectroscopy on single liposomes and single cells: (i) target membranes and cells will be bound to the planar viral spike glycoprotein-containing membranes; (ii) chemical cross-linking, resonance energy transfer and lateral diffusion experiments will be performed in the supported membrane-target membrane contact region; and (iii) the membrane-membrane contact regions will be characterized by digital video microscopy. In addition, and to accomplish aim (4), (i) the fusion peptide of HA2 will be selectively changed by oligonucleotide-directed mutagenesis and analyzed for its fusion caoacity and (ii) corresponding fusion peptides will be chemically synthesized and their secondary structure, orientation and depth of membrane-penetration will be determined in phospholipid bilayers. Taken together, these studies will provide a structural and functional basis for understanding the mechanism of viral spike glycoprotein-mediated membrane fusion.